The majority of solid tumors exhibit both structural and numerical chromosome aberrations characteristic of genomic instability. Chromosome instability (CIN), the frequent and persistent gains and losses of whole chromosomes results in the generation of aneuploid cells and has important implications. CIN has been proposed to facilitate the evolution of tumor cells by promoting genetic heterogeneity, thereby enabling changes that promote growth and metastasis. Furthermore, genomic diversity generated by CIN promotes the development of cancer cells that are resistant to therapeutics and are more prone to tumor relapse. Consequently, CIN correlates with poor patient prognosis. Limitations in identifying and treating cancers that exhibit CIN stem from a lack of understanding of the underlying molecular mechanisms that contribute to CIN and the inability to manipulate CIN in a therapeutically relevant manner. I have recently demonstrated that pRB, a tumor suppressor whose function is compromised in many human cancers, promotes genome stability. I have shown that pRB loss leads to defects in chromatin structure, and that these abnormalities promote increased DNA damage and corruption of mitotic fidelity. However, corruption of the pRB pathway is not the only mechanism by which chromatin structure is compromised, and other regulators of chromatin structure are likely to similarly impact genome stability. One manner in which chromatin structure is impacted is through regulation of chromatin and histone methylation status. Indeed, a growing body of evidence has suggested that modification of epigenetic characteristics of cells correlates with aneuploidy. In addition, my preliminary studies have shown that modulation of levels of a subset of methyl-transferases can compromise chromatin structure and genome integrity. Together, these data suggest that general deregulation of chromatin structure is broadly relevant to the genesis of genomic instability. However, this has not been analyzed in a systematic manner, and the mechanisms by which chromatin structure are deregulated and contributes to genome instability remain unknown. Significantly, I have shown that by restoring chromosomal structure, I can suppress both DNA damage and promote accurate chromosome segregation in tumor cells. In doing so, I have identified Wapl, a negative regulator of cohesion as a novel regulator of CIN. In the following proposal, this novel strategy will enable me to test, for the first time, whether suppression of CI renders tumor cells less capable of developing chemotherapeutic resistance in vivo, and thus less likely to relapse following traditional treatments. In addition, using a combination of bioinformatics and cell biological approaches, I will examine whether structural defects associated with CIN can be exploited to increase sensitivity to DNA damaging agents and decrease cell survival. Finally, I will identify regulators of chromatin structure whose deregulation impacts genome stability using a novel screening approach I have developed. The successful completion of the aims described in this proposal will not only provide new and valuable insights into the genesis of CIN in human cancers, but also reveal the therapeutic ramifications of manipulating this common feature in tumors. My career goal is to obtain a research faculty position at a leading institute where I will define the tumor suppressive mechanisms that maintain normal chromosome structure and genomic integrity. However, my successful transition to independence in this field would be significantly bolstered by augmenting my expertise in cell biological techniques with new training opportunities in mouse models of cancer, bioinformatics and high throughput screening approaches. It is with these acquired skills that I will be able to identify cancer-relevant regulators of chromatin structure tat impact genome stability and assess their roles as novel tumor suppressors. The success of this project will be greatly enhanced by the outstanding training committee that I have assembled to mentor and advise me as I progress towards independence. This committee includes my co-mentors Dr. Nicholas Dyson and Dr. Jeffrey Engelman, as well as Dr. Daniel Haber, Dr. Cyril Benes, and Dr. David Pellman, all of whom are experts in their respective fields. In addition, the exceptional research environment available at MGH and the Harvard Medical Area has all of the necessary physical resources required for the completion of the proposed training and research studies. The K99/R00 award would afford me the protected time needed for this advanced training and allow me to continue to foster my growth under the mentorship of Dr. Dyson and Dr. Engelman. I expect that the mentored phase of this proposal, which includes using mouse models to investigate the therapeutic relevance of manipulating CIN in solid tumors, and the initiation of a novel screening approach, will take 1-2 years and result in at least one major publication. The following independent phase of the award will allow me to identify and characterize additional, cancer-relevant, regulators of chromosome structure and genome stability. Together these data will be used to justify future studies proposed in an R01 grant that I plan to submit at the start of the third year of the independent phase.